CN109762062A - A kind of preparation method of goose goat Yolk antibody - Google Patents
A kind of preparation method of goose goat Yolk antibody Download PDFInfo
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Abstract
A kind of preparation method of goose goat Yolk antibody.Present invention RPVA1061 plants of isolated goose astrovirus from the young goose pathologic liver and kidney of clinical infection, prevention and treatment of the Yolk antibody for clinical young bird goose goat is prepared into using RPVA1061 plants, while solving the problems, such as existing clinical goose goat without drug, provide a kind of clinical prevention scheme of new goose goat, according to clinical verification, the Yolk antibody of preparation of the invention is significant to the control efficiency of goose goat.
Description
Technical field
The invention belongs to Yolk antibody preparation technical fields, and in particular to a kind for the treatment of goose gout as caused by astrovirus
The preparation method of the Yolk antibody of disease.
Background technique
Year-to-date from 2017, in the young gaggle on the ground such as China Shandong, Jiangsu, Guangdong, Guangxi, Hunan, Henan and Anhui
Occur a kind of using internal organ and arthragra as the communicable disease of cardinal symptom, supports goose industry to China and cause serious financial consequences.
The disease takes place mostly in the young goose of 1-20 age in days, and the death rate reaches as high as 60%.Illness young bird goose spirit is depressed, sleepingly tired dynamic, adopts
Food is reduced.Dissect shows as the serious uric acid mineralization of internal organs and articular cavity, and different cultivars uses different feeds, difference
The gaggle of drug has generation, and the protein content, reduction scale of feeding in reduction feed are invalid.After the disease occurs, we are from wide
The goose field that gout occurs for the provinces such as east, Hunan, Anhui, Henan acquires more than 100 part of pathological material of disease, has carried out cause of disease point to the pathological material of disease of acquisition
From with animal Orthogonal Rotational Regressive Tests, determining causes the cause of disease of the disease for novel goose astrovirus.
Before this, in industry by goose goat because key factor is in feed nutrient is unbalance and drug abuse, for
The prevention and treatment of goose gout virus, at present other than reinforcing disinfection and improving goose field cultivating condition, it is effective that there is no other
Means.On the basis of verifying the cause of disease, the means of prevention of virosis, vaccine immunity and antibody note are directed in conjunction with existing cultivation industry
Penetrating treatment is most effective and most economical means of prevention, since vaccine immunity is there are the Blank immunization phase, is not suitable for goose goat
Characteristics of incidence, therefore Antybody therapy becomes the control measure of prevention and treatment goose goat unique feasible.Yolk antibody is because having antibody system
The advantages that standby economic, easy to operate, yield is big, potency is high, therefore the Yolk antibody for goose astrovirus is prepared as current
Prevent and treat the disease need and it is preferred.
Summary of the invention
The purpose of the present invention is to provide a kind of clinical prevention schemes of new goose goat.
Of the invention also residing in provides a kind of Yolk antibody for preventing and treating goose goat, to solve in existing clinic for goose pain
The problem of wind disease can be used without medicine.
Of the invention also residing in provides a kind of preparation method of Yolk antibody for preventing and treating goose goat.
To realize the above-mentioned technical purpose, the present invention adopts the following technical scheme:
A kind of preparation method of goose goat Yolk antibody, uses the deposit number of inactivation for CCTCCNo.V201839's
Goose astrovirus Immune Laying Hens, then the extraction purification Yolk antibody from egg yolk.
The specific preparation process of goose goat Yolk antibody be the following steps are included:
1) goose astrovirus is separated from goose gout clinical case, and star is determined that it is by PCR identification and Serologic detection
Shape virus；
2) by above-mentioned isolated goose astrovirus through the susceptible goose embryo preparation production poison of allantocherion vaccination；
3) it is produced with formalin-inactivated with after poison, is prepared into inactivated vaccine；
4) inactivated vaccine Immune Laying Hens are used, height is collected and exempts from the isolated goose astrovirus Yolk antibody of egg yolk.
The goose astrovirus be it is isolated from the young goose pathologic liver and kidney of clinical infection, named
It is RPVA1061 plants, which is preserved in China typical culture collection center (CCTCC), preservation on June 28th, 2018
Number are as follows: CCTCC No.V201839, depositary institution address are as follows: Wuhan City, Hubei Province Wuchang District Bayi Road 299.
It is described production with poison be from the allantoic fluid, amniotic fluid and idiosome of susceptible goose embryo dead after inoculation, homogenate, freeze thawing,
Be collected by centrifugation, be concentrated by ultrafiltration after obtain.
The formalin-inactivated of the production poison refers to: the formaldehyde of final concentration of 0.08-0.25% is added in production poison,
30-42 DEG C inactivation 18-24 hours.
Further, using the formaldehyde of final concentration 0.1%, in 37 DEG C of inactivation productions with poison 18 hours.
The inactivated vaccine is oil emulsion inactivated vaccine.
Further, the oil of the inactivated vaccine is mutually white oil.
The Immune Laying Hens at least need to carry out a fundamental immunity and twice booster immunization.
Further, the Immune Laying Hens carry out booster immunization three times.
The height exempts to start within 14 days collection after egg refers to last time booster immunization.
The separation of the goose astrovirus Yolk antibody answer comprising the following steps:
(1) eggshell sterilizes: high-immunity egg being immersed to soaking disinfection 15min in 0.1% bromogeramine solution, after taking out disinfection
Egg naturally dry or drying, spray 75% alcohol it is after disinfection to reserve to eggshell surface.
(2) yolk separation takes machinery to beat eggs: sufficiently removing egg white, blastodisc and frenulum when beating eggs, collects yolk.
(3) it inactivates I: the yolk of collection is sufficiently stirred, make yolk in uniform paste, start peristaltic pump, by yolk liquid pump
Enter in interlayer reactor tank, is added and the isometric water for injection of yolk, after stirring and evenly mixing, 60~65 DEG C of heat preservation (inactivation) 30min.
(4) acidification extracts: the acetic acid for the pH value 5.0 that first addition is equivalent to former 3 times of volumes of yolk in isolation reactor tank is slow
Then yolk liquid is added in fliud flushing, open blender and stir, abundant agitating 30 minutes.
(5) it inactivates II: the octanoic acid that final concentration (V/V) is 4% being added in yolk liquid liquid and makees inactivator and extracts agent, acutely stirs
It mixes 90 minutes, 2~8 DEG C are placed 4~8 hours.
(6) it coarse filtration: is filtered until clear with after polypropylene fibre 750B filter-cloth filtering, then with column core filter.
(7) aseptic filtration: with 0.22 μm of micropore core filtration sterilization, setting 2~8 DEG C of storages, should be no more than 14.
(8) it inactivates III: filtered solution is imported in inactivation tank, metered 10% formalin opens stirring
Machine stirring, mixes them thoroughly, and the ultimate density (V/V) of formalin is 0.1%, and 37 DEG C inactivate 16 hours to obtain the final product.
A kind of antigen being used to prepare goose goat Yolk antibody, the antigen are RPVA1061 plants of goose astrovirus, are protected
Hiding number are as follows: CCTCC No.V201839.
Further, the antigen is deposit number are as follows: CCTCC No.V201839 is through PCR and Serologic detection
After be determined as astrovirus.
The invention has the beneficial effects that:
1) it overcomes in existing clinic and the cause of disease of goose goat is attributed to that feed nutrient is unbalance and drug abuse
It is related with goose astrovirus to explicitly point out the morbidity of goose goat for technology prejudice；
2) it is that applicant oneself is isolated, and incites somebody to action that deposit number, which is the goose astrovirus strain of CCTCC No.V201839,
It is prepared into Yolk antibody and solves the problems, such as to use in existing clinic without medicine；
3) the goose astrovirus Yolk antibody potency height of preparation, good immune effect, can effectively prevent goose goat, enhance machine
Body immunity function escorts for healthy aquaculture；
4) goose goat Yolk antibody preparation process is simple, high-efficient, production cost is low.
Detailed description of the invention
Fig. 1 .PCR identifies goose gout gel electrophoresis analysis figure
Fig. 2 goose astrovirus animal returns dissect result
Fig. 1 explanation: being positive control from the marker label right side 1, goose gout pathological material of disease of the right side 2 to be identified, the right side 3 is negative right
According to.
Fig. 2 explanation: upper left gollbladder dilation, bile is full in emerald green, there is uric acid salt particle, upper right heart surface in bile
There are uratic deposit, the pale enlargement of lower-left kidney, bottom right heart has lithate package.
Specific embodiment
Detailed description below is all illustrative, it is intended to provide further instruction to the present invention.Unless otherwise indicated, originally
All scientific and technical terms that text uses have and the normally understood identical meanings of the technical field of the invention personnel.
The separation of the wild goose astrovirus of embodiment 1 is identified
By from clinical infection young goose pathologic liver and kidney homogenized in non-viral delivery medium with antibiotic, by this
The tissue that homogenizes is filtered by 0.45 micron syringe filter.The homogenate of 0.1ml virus liquid is used on primary goose embryonic kidney cells
0.1-2% serum continuously cultivated for 4 generations, groped to appropriate virus serum free culture system concentration, about 0.5-1%.With 70%-80% cell
Cell culture is freezed and is stored at -80 DEG C by the development of pathological effect.
(1) RNA is extracted.Using RNA/DNA Viral extraction kit (Takara), to the young goose pathologic liver of clinical infection
And primary goose embryonic kidney cells culture carries out viral RNA extraction.
(2) design of primers.It is used using published goose astrovirus sequence according to the ORF1b sequence that astrovirus is guarded
Primer5.0 software carries out design of primers.Primer sequence design is as follows:
F:GACTGGACAAGCTACGATGGCACTATTCC (SEQ ID No.1)
R:CTTAACCCACATGCCGAA (SEQ ID No.2)
ORF1b sequence is referring to sequence table: SEQ ID No.3.
(3)RT-PCR.Viral RNA extraction is carried out using virus RNA extraction kit (Takara), uses reverse transcriptase
(Takara) reverse transcription is carried out.
Specific step is as follows for reverse transcription:
1) following template ribonucleic acid/primer mixed liquor, 6 μ l of full dose are prepared in Microtube pipe
The μ of template ribonucleic acid 1ng~1 g
Oligo(dT)12-18Primer(50μM) 1μl
RNase free H2O up to 6μl
2) extremely cold 5min on ice after 70 DEG C of heat preservation 10min, it is short from.
3) following reaction solution is added in above-mentioned Microtube pipe
4) 42 DEG C of heat preservation 1h
5) cooled on ice after 70 DEG C of heat preservation 15min, obtains cDNA solution.
PCR is carried out using taq enzyme (Takara), specific PCR reaction condition is as follows:
It carries out DNA gel electrophoretic analysis (1% gel), obtains the target stripe of 440bp, be consistent with expected clip size,
The result is shown in Figure 1 send Huada gene company to be sequenced after purpose band recovery purifying, and will disclose on sequencing result and GenBank
The conservative ORF1b sequence of goose astrovirus be compared, homology 99.6%, it was demonstrated that there are goose astrovirus in pathological material of disease.
The passage of goose astrovirus goose embryo:
To die of illness young Goose Liver and renal tissue, and sterile phosphate buffer is added according to the ratio of 1:4 and carries out homogenized,
Equal slurries are placed in -20 DEG C of refrigerators to room temperature multigelation 2 times, 8000rpm takes supernatant after being centrifuged 20min, through 0.22 μm of needle
- 20 DEG C of preservations after formula filter filtration sterilization.
The tissue poisons chorioallantoic membrane approach 10 age in days goose embryos of inoculation that will acquire, 0.2ml/ pieces, and it is slow with Sterile phosphate
Negative control is arranged in fliud flushing, and 30 DEG C of incubations, inspection egg 2 times, discard goose embryo dead within 24 hours, be observed continuously 7 daily, will
Dead goose embryo sets sterile collection allantoic fluid and the obvious embryo of lesion after 4 DEG C of cold eggs, 4 generation of venom blind passage.
Goose astrovirus target animals return:
The young goose for taking 30 1 ages in days, is randomly divided into 2 groups.The subcutaneously virus inoculation allantoic fluid dosage of inoculation of I group 20
0.2ml/ is only；II group 10 is in the same way with dose inoculation sterile saline as control.It is daily to observe facing for young goose
Bed symptom and death condition.Dead young goose dissect on the 3rd has typical young goose gout symptom after attacking poison: the pale enlargement of kidney, heart have
Uric acid mineralization, gall-bladder fill bile has uric acid salt particle to precipitate (as shown in Figure 2) in emerald green.
The astrovirus of above-mentioned separation identification is RPVA1061 plants of goose astrovirus, deposit number are as follows: CCTCC
No.V201839。
The preparation of 2 goose astrovirus venom of embodiment and inactivated vaccine
1, expand poison: RPVA1601 plants of seeds culture of viruses are made 100 times and are diluted, 100 pieces of the susceptible goose embryo of 10 age in days of allantocherion vaccination, often
Embryo 0.2ml, 36~37 DEG C of incubations, 2 times a day according to embryo inspection.Goose embryo dead in 48~240 hours after inoculation, sets 2~8 DEG C and puts
After setting 4~12 hours, allantoic fluid, amniotic fluid and idiosome are collected, idiosome goes to turn around and four limbs.After being homogenized with blastochyle, freeze thawing 3 times,
8000r/min is centrifuged 20min, takes supernatant to be mixed in sterile chamber, sets 2~8 DEG C of preservations.(referring to table 1).
The preparation of 1 venom of table
| Strain name | Embryo kind | It is inoculated with embryo number (piece) | Embryo age in days | It harvests venom (ml) |
| RPVA1601 | Susceptible goose embryo | 100 | 10 | 1480 |
2, it is concentrated: by the RPVA strain virus liquid of harvest under the conditions of 2~8 DEG C, being concentrated into substance respectively with ultrafiltration concentration machine
Long-pending 1/4 carries out steriling test, asepsis growth by existing " Chinese veterinary pharmacopoeia " annex, and the malicious valence of standby survey that keeps sample.RPVA strain virus
The every 0.2ml viral level of liquid answers >=105.5ELD50, the blastochyle after concentration inactivated immediately.(referring to table 2).
The concentration of 2 venom of table
3, it inactivates: the RPVA1601 strain virus liquid after concentration being imported in inactivation tank, 10% formalin is added, fills it
Divide mixing, the ultimate density of formalin is 0.1%.It is imported in another inactivation tank after adding formalin, near tank mouth
The virus of adherency fails to contact inactivator.37 DEG C of sealings inactivate 18 hours (by temperature in tank reach 37 DEG C start in terms of), therebetween often
It is primary every stirring in 4~6 hours, inactivate 2~8 DEG C of postposition preservations.
4, prepared by inactivated vaccine:
(1) oil is mutually prepared: 94 parts of white oil for animals are taken, 2 parts of aluminum stearate, is placed in oily phase preparation tank after being heated to 80 DEG C,
6 parts of Jia Siben -80 again, until 30min is maintained when temperature reaches 115 DEG C, it is spare after cooling.
(2) prepared by water phase: inactivation being examined to qualified 96 parts of RPVA1601 strain additions, 4 parts of sterilizing Tween-80s, starts stirring
Motor stirs 20~30min, is completely dissolved Tween-80.
(3) emulsify: it is 2:3 (V/V) that water phase and oil, which mix emulsification ratio, first by oily mutually importing colloid mill, 2500 revs/min
Clock stirring, is slowly added to water phase, emulsifies 5 minutes for 10000 revs/min after adding.Vaccine total amount 0.01% is pressed before terminating emulsification
Thimerosal is added, sufficiently oscillation mixes.After emulsification, 10ml is taken, with 3000r/min centrifugation 15 minutes, tube bottom was precipitated without water phase.
(4) it after aseptic subpackaged, is saved after rolling lid at 2~8 DEG C.(referring to table 3)
The emulsification of 3 vaccine of table and packing
The preparation of 3 goose astrovirus Yolk antibody of embodiment
1, the preparation of high-immunity egg: by the goose astrovirus inactivated vaccine Immune Laying Hens of above-mentioned preparation, every chicken of first immunisation
2.0ml inactivated vaccine is subcutaneously injected in neck, and second is carried out after 21 days and is immunized, and every chicken neck subcutaneous injection 2.0ml inactivates epidemic disease
Seedling, two, which exempt from latter 21 days progress third times, is immunized, and 2.0ml inactivated vaccine is subcutaneously injected in every chicken neck, and three exempt from rear added for 21 days
It is immunized by force, every chicken neck subcutaneous injection 2.0ml inactivated vaccine, 14 days after booster immunization, acquisition yolk carries out attacking malicious protection
Experiment.
2, Yolk antibody manufactures:
(1) eggshell sterilizes: 18 kilograms of high-immunity eggs being immersed to soaking disinfection 15min in 0.1% bromogeramine solution, are taken out
Egg naturally dry or drying after disinfection, spray 75% alcohol it is after disinfection to reserve to eggshell surface.
(2) yolk separation takes machinery to beat eggs: sufficiently removing egg white, blastodisc and frenulum when beating eggs, collects yolk.
(3) it inactivates I: the yolk of collection is sufficiently stirred, make yolk in uniform paste, start peristaltic pump, by yolk liquid pump
Enter in interlayer reactor tank, (water for injection is first sterilized through 80 DEG C of 30min, and is cooled to the isometric water for injection of yolk for addition
65 DEG C or less), after stirring and evenly mixing, 60~65 DEG C of heat preservation (inactivation) 30min.
(4) acidification extracts: the acetic acid for the pH value 5.0 that first addition is equivalent to former 3 times of volumes of yolk in isolation reactor tank is slow
Then yolk liquid is added in fliud flushing, open blender and stir, abundant agitating 30 minutes.
(5) it inactivates II: the octanoic acid that final concentration (V/V) is 4% being added in yolk liquid liquid and makees inactivator and extracts agent, acutely stirs
It mixes 90 minutes, 2~8 DEG C are placed 4~8 hours.
(6) it coarse filtration: is filtered until clear with after polypropylene fibre 750B filter-cloth filtering, then with column core filter.
(7) aseptic filtration: with 0.22 μm of micropore core filtration sterilization.2~8 DEG C of storages are set, should be no more than 14.
(8) it inactivates III: filtered solution is imported in inactivation tank, metered 10% formalin opens stirring
Machine stirring, mixes them thoroughly, and the ultimate density (V/V) of formalin is 0.1%, and 37 DEG C inactivate 16 hours.
Specific production target and it the results are shown in Table 4:
The manufacture of 4 Yolk antibody of table and packing
4 goose astrovirus Yolk antibody of embodiment attacks malicious protecting effect
With the susceptible young goose 30 of 1~5 age in days health, it is randomly divided into 3 groups, every group 10.1st group is healthy control group, no
Inject any drug, independent isolated rearing；2nd group to attack malicious control group, every subcutaneously or intramuscularly injecting normal saline 0.5ml；
3rd group is Yolk antibody group, and every is subcutaneously or intramuscularly injected Yolk antibody 0.5ml.The 7th day after injection, with goose astrovirus
RPVA1601 strain virus liquid is subcutaneously injected the 2nd group and the 3rd group, every 1.0ml (containing 100ID50).Observation 10 days.1st group should be complete
Portion is strong to live；2nd group should at least fall ill 8 and test and can set up.
Attack malicious group after attacking poison third day start to fall ill, observation terminates to share 9 morbidities to experiment, disease incidence 90%, in advance
Anti- group has 1 morbidity for the 5th day after attacking poison, and observation to experiment terminates to share 1 morbidity, and prevention & protection rate is 90%, and attacks poison
There were significant differences for control group, illustrates that young goose gout Yolk antibody can effectively prevention & protection goose astrovirus attack.
The above is merely a preferred embodiment of the present invention, it is noted that the those of ordinary skill in this technology is come
It says, under the premise of not departing from core of the invention technical characteristic, several improvements and modifications can also be made, these are retouched and change
Into also should belong to scope of patent protection of the invention.
Claims (10)
1. a kind of preparation method of goose goat Yolk antibody, it is characterised in that: use the deposit number of inactivation for CCTCC
The goose astrovirus Immune Laying Hens of No.V201839, then the extraction purification Yolk antibody from egg yolk.
2. Yolk antibody preparation method according to claim 1, it is characterised in that: the preparation method packet of the Yolk antibody
Include following steps:
1) it separates, detect from goose gout clinical case, identification goose astrovirus；
2) by isolated goose astrovirus through the susceptible goose embryo preparation production poison of allantocherion vaccination；
3) it is produced with formalin-inactivated with after poison, is prepared into inactivated vaccine；
4) inactivated vaccine Immune Laying Hens are used, height is collected and exempts from the isolated goose astrovirus Yolk antibody of egg yolk.
3. Yolk antibody preparation method according to claim 2, it is characterised in that: the formalin-inactivated, which refers to, to be produced
With the formaldehyde that final concentration of 0.08-0.25% is added in poison, 30-42 DEG C inactivation 18-24 hours.
4. Yolk antibody preparation method according to claim 2 or 3, it is characterised in that: the formalin-inactivated refers to
Using the formaldehyde of final concentration 0.1%, in 37 DEG C of inactivation productions with poison 18 hours.
5. Yolk antibody preparation method according to claim 2, it is characterised in that: the Immune Laying Hens, at least need into
Fundamental immunity of row and twice booster immunization.
6. Yolk antibody preparation method according to claim 2 or 5, it is characterised in that: the Immune Laying Hens need to carry out
Booster immunization three times.
7. Yolk antibody preparation method according to claim 2, it is characterised in that: the height exempts from egg and refers to last time
Start within 14 days after booster immunization the egg collected.
8. Yolk antibody preparation method according to claim 2, it is characterised in that: isolated goose astrovirus yolk is anti-
Body comprising the following steps:
1) exempt from egg to height to carry out disinfection；
2) egg white and yolk are separated；
3) after the vitellinae membrana and frenulum of place to go yolk, yolk liquid is prepared into colloid mill is broken；
4) acidification is extracted and is inactivated；
5) clear filtrate is collected by filtration up to Yolk antibody.
9. Yolk antibody preparation method according to claim 8, it is characterised in that: the inactivation should include 60-65 DEG C
Heat preservation inactivates the formalin-inactivated with final concentration of 0.1%.
10. a kind of goose astrovirus for being used to prepare Yolk antibody, it is characterised in that: the astrovirus is from clinical infection
Young goose pathologic liver and kidney in RPVA1061 plants isolated, deposit number are as follows: CCTCC No.V201839.
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| CN113214388A (en) * | 2021-05-12 | 2021-08-06 | 北京世华康源生物科技有限公司 | Goose astrovirus egg yolk antibody and preparation method thereof |
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| CN110251671A (en) * | 2019-06-28 | 2019-09-20 | 重庆永健生物技术有限责任公司 | Goose astrovirus Yolk antibody compound and preparation method thereof |
| CN110251671B (en) * | 2019-06-28 | 2023-02-24 | 重庆永健生物技术有限责任公司 | Preparation method of goose astrovirus egg yolk antigen-antibody complex |
| CN113214388A (en) * | 2021-05-12 | 2021-08-06 | 北京世华康源生物科技有限公司 | Goose astrovirus egg yolk antibody and preparation method thereof |
| CN113214388B (en) * | 2021-05-12 | 2022-06-28 | 南阳师范学院 | Goose star virus egg yolk antibody and preparation method thereof |
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| CN109762062B (en) | 2022-02-25 |
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